Mucosal HIV-1 transmission accounts for the majority of new HIV infections, yet is a poorly understood event. Clinical and experimental evidence supports the role of local immune activation and inflammation in facilitating HIV-1 transmission. The immunologic factors contributing to this phenomenon are an area of intense interest. The intestinal microbiome consists of a diverse collection of organisms, many of which influence mucosal inflammation and immunity. Alterations in the microbiome, termed dysbiosis, have been associated with HIV-1 infection, yet the drivers of these dysbiosis and its effect on HIV susceptibility remain unclear. Substance use is known to increase risk of HIV-1 acquisition and alter the gut microbiome, and its study therefore offers an opportunity to examine a mechanistic link between a defined environmental factor and HIV-1 transmission through effects on the microbiome and/or mucosal immunity. We hypothesize that increased HIV-1 acquisition risk due to substance use results from pro-inflammatory alterations of the intestinal microbiome and/or direct effects on the inflammatory state of the mucosa, thereby increasing HIV-1 susceptibility. This proposal aims to investigate this hypothesis by characterizing the intestinal microbiome using a unique cohort of young men who have sex with men (MSM) who are HIV-1 seropositive or at-risk. Our study has the unique advantage of specimens coupled with detailed clinical, substance use, and sexual behavior data which will allow us to address influence of these factors on dysbiosis and mucosal inflammation. Using these specimens, we can employ novel ex vivo assays to directly examine the mechanisms mediating microbiome effects on mucosal immune activation and HIV-1 susceptibility. Specifically, we propose to: 1) characterize the impact of risk factors such as substance use on the intestinal microbiome in HIV-positive and at-risk individuals, 2) examine the mucosal activation status and its relationship to dysbiosis in at-risk individuals, and 3) optimize mucosal HIV-1 infection assays to quantitate susceptibility of gut mucosa. These findings may reveal potentially modifiable targets to improve HIV-1 prevention strategies in certain at-risk populations. This career development award application will support the training, mentoring, and research of a promising junior investigator. The environment offers the combination of exceptional faculty and staff, state-of-the-art resources and facilities, and an institutional commitment to research which all serve to foster the success of this research project and career development plan. The proposed research will allow the applicant to receive specific advanced training in the following focused areas: 1) bioinformatics and computational methods, 2) advanced statistics, 3) mucosal immunology and microbiology, and 4) academic scientific research. The training plan includes a combination of directed didactic training and mentoring by leading scientists while conducting the proposed research. These skills will not only advance the goals of the proposed project, but also provide a foundation to build a lasting independent career as a successful physician-scientist.